The objective of this proposal is to develop a robust, high throughput, cell-based assay of Salmonella infection. In the US, 2-4 million Salmonella infections occur annually, which account for nearly 30% of all deaths from food-borne disease. Widespread Salmonella infections of livestock and other animals also have important public health implications. During infection, Salmonella passes through the intestinal wall by adhering to the surface of epithelial cells and inducing the formation of membrane ruffles. These protrusions extend around the bacterium, engulfing it and taking it into the cell. Using a needle-like structure, called a type III secretion system, Salmonella injects a battery of effector proteins into the host cell. Some of these proteins are able to mimic host cell activators of actin polymerization, resulting in membrane ruffling and macropinocytosis. The objective of this proposal is to develop a high throughput screen to quantify Salmonella invasion, which can be used to interrogate small molecule libraries to identify inhibitors of Salmonella host cell invasion. We have developed a cell-based assay in a microtiter plate, which uses luciferase to identify Salmonella invasion. The Specific Aims are: 1) Optimize a cell-based assay to quantify Salmonella infection in a 96-well plate or higher density format. 2) Configure the assay for high throughput screening. This will include performing pilot screens and evaluating positive screen hits for mammalian and bacterial toxicity, followed by secondary, tertiary and counter screens. Once the assay is optimally configured, we intend to submit it to MLPCN for implementation. This high throughput screen can be used to identify small molecule inhibitors of Salmonella invasion. Moreover, the assay principle could potentially be applied to almost any organism that invades humans or animals. These molecular probes will be valuable reagents to advance our understanding of the cellular mechanism of microbial pathogenesis. Identification of useful biological targets could ultimately result in the development of novel therapies to prevent and/or treat salmonellosis. PUBLIC HEALTH RELEVANCE: Infection of both humans and animals by Salmonella is a major cause of morbidity and mortality. We have developed a cell-based assay in a microtiter plate to identify Salmonella invasion. The objective of this proposal is to optimize the assay in a high throughput format.